Search

Spinal arteriovenous malformation (AVM) is a rare disease that arises more commonly from the lower thoracic and upper lumbar spine. It is classified based on the development pattern and the shunt location. The developmental mechanism of the symptoms is thought to be as follows. Spinal venous return is impaired by high-pressure arterial blood flowing into the coronary sinus via a shunt and venous pressure is promoted, which causes spinal cord symptoms to progress gradually. Listlessness, pain, and an abnormal sensation of the lower limb are possible initial symptoms. Spinal AVM may cause intermittent claudication and bladder and rectal disturbance, and differentiation from lumbar degenerative diseases and arteriosclerosis obliterans is required, which may cause difficulty with diagnosis. We encountered a patient in whom intermittent claudication was treated with lumbar decompression and fixation at another hospital, but symptoms did not improve. The patient was diagnosed with spinal AVM at our hospital and symptoms were improved by surgery. Because the symptoms did not improve despite being treated with surgery for spinal canal stenosis, we strongly suspected spinal intermittent claudication, and we performed a spinal CT and MRI after myelography for the entire spinal cord, and identified the lesion in the thoracic spinal cord. Regarding the postoperative outcome and prognosis of spinal dural AVF, early diagnosis and early treatment have been proposed as prognostic factors because the postoperative outcome is poor in patients with high preoperative severity and a long duration of illness. In our patient, the preoperative JOA score was 6, showing high preoperative severity, and the duration of illness was 1.5 years before diagnosis, which may explain the limited improvement of the JOA score to 20 at final follow-up. In a case with these characteristics, we suggest that close examination of the entire spinal cord is needed for effective treatment.

Polysialic acid (polySia) is a linear polymer of sialic acid that modifies neural cell adhesion molecule (NCAM) in the vertebrate brain. PolySia is a large and exclusive molecule that functions as a negative regulator of cell-cell interactions. Recently, we demonstrated that polySia can specifically bind fibroblast growth factor 2 (FGF2) and BDNF; however, the protective effects of polySia on the proteolytic cleavage of these proteins remain unknown, although heparin/heparan sulfate has been shown to impair the cleavage of FGF2 by trypsin. Here, we analyzed the protective effects of polySia on the proteolytic cleavage of FGF2 and proBDNF/BDNF. We found that polySia protected intact FGF2 from tryptic activity via the specific binding of extended polySia chains on NCAM to FGF2. Oligo/polySia also functioned to impair the processing of proBDNF by plasmin via binding of oligo/polySia chains on NCAM. In addition, the polySia structure synthesized by mutated polysialyltransferase, ST8SIA2/STX(SNP7), which was previously identified from a schizophrenia patient, was impaired for these functions compared with polySia produced by normal ST8SIA2. Taken together, these data suggest that the protective effects of polySia toward FGF2 and proBDNF may be involved in the regulation of the concentrations of these neurologically active molecules.

The BRCT repeats of the breast and ovarian cancer predisposition protein BRCA1 are essential for tumor suppression. Phosphopeptide affinity proteomic analysis identified a protein, Abraxas, that directly binds the BRCA1 BRCT repeats through a phospho-Ser-X-X-Phe motif. Abraxas binds BRCA1 to the mutual exclusion of BACH1 (BRCA1-associated C-terminal helicase) and CtIP (CtBP-interacting protein), forming a third type of BRCA1 complex. Abraxas recruits the ubiquitin-interacting motif (UIM)–containing protein RAP80 to BRCA1. Both Abraxas and RAP80 were required for DNA damage resistance, G 2 -M checkpoint control, and DNA repair. RAP80 was required for optimal accumulation of BRCA1 on damaged DNA (foci) in response to ionizing radiation, and the UIM domains alone were capable of foci formation. The RAP80-Abraxas complex may help recruit BRCA1 to DNA damage sites in part through recognition of ubiquitinated proteins.

SummaryFanconi anemia (FA) is a developmental and cancer-predisposition syndrome caused by mutations in genes controlling DNA interstrand crosslink repair. Several FA proteins form a ubiquitin ligase that controls monoubiquitination of the FANCD2 protein in an ATR-dependent manner. Here we describe the FA protein FANCI, identified as an ATM/ATR kinase substrate required for resistance to mitomycin C. FANCI shares sequence similarity with FANCD2, likely evolving from a common ancestral gene. The FANCI protein associates with FANCD2 and, together, as the FANCI-FANCD2 (ID) complex, localize to chromatin in response to DNA damage. Like FANCD2, FANCI is monoubiquitinated and unexpectedly, ubiquitination of each protein is important for the maintenance of ubiquitin on the other, indicating the existence of a dual ubiquitin-locking mechanism required for ID complex function. Mutation in FANCI is responsible for loss of a functional FA pathway in a patient with Fanconi anemia complementation group I.

The evolutionarily conserved Hus1 proteins function in DNA damage response pathways that serve to maintain genomic stability [1, 2]. Cells lacking mouse Hus1 are hypersensitive to certain genotoxins [3], and we have explored the molecular basis for this defect by examining how Hus1 inactivation affects genotoxin-induced signaling events. p53 accumulation and activation in response to DNA damage appeared normal in Hus1 null cells. Likewise, Hus1 was dispensable for genotoxin-induced Chk2 phosphorylation. In contrast, Chk1 phosphorylation after genotoxic stress was greatly reduced in the absence of Hus1, but was restored in Hus1 null fibroblasts complemented by infection with a Hus1 -expressing retrovirus. These results demonstrate that mouse Hus1 is required for a specific subset of DNA damage signaling events and functions to promote genotoxin-induced Chk1 phosphorylation.

Chk1, an evolutionarily conserved protein kinase, has been implicated in cell cycle checkpoint control in lower eukaryotes. By gene disruption, we show that CHK1 deficiency results in a severe proliferation defect and death in embryonic stem (ES) cells, and peri-implantation embryonic lethality in mice. Through analysis of a conditional CHK1-deficient cell line, we demonstrate that ES cells lacking Chk1 have a defective G2/M DNA damage checkpoint in response to γ-irradiation (IR). CHK1heterozygosity modestly enhances the tumorigenesis phenotype ofWNT-1 transgenic mice. We show that in human cells, Chk1 is phosphorylated on serine 345 (S345) in response to UV, IR, and hydroxyurea (HU). Overexpression of wild-type Atr enhances, whereas overexpression of the kinase-defective mutant Atr inhibits S345 phosphorylation of Chk1 induced by UV treatment. Taken together, these data indicate that Chk1 plays an essential role in the mammalian DNA damage checkpoint, embryonic development, and tumor suppression, and that Atr regulates Chk1.

Cyclin-dependent kinases (Cdks) are positive regulators of cell proliferation, whereas Cdk inhibitors (CKIs) inhibit proliferation. We describe a new CKI, p57KIP2, which is related to p21CIP1 and p27KIP1. p57KIP2 is a potent, tight-binding inhibitor of several G1 cyclin/Cdk complexes, and its binding is cyclin dependent. Unlike CIP1, KIP2 is not regulated by p53. Overexpression of p57KIP2 arrests cells in G1. p57KIP2 proteins have a complex structure. Mouse p57KIP2 consists of four structurally distinct domains: an amino-terminal Cdk inhibitory domain, a proline-rich domain, an acidic-repeat region, and a carboxy-terminal domain conserved with p27KIP1. Human p57KIP2 appears to have conserved the amino- and carboxy-terminal domains but has replaced the internal regions with sequences containing proline-alanine repeats. In situ hybridization during mouse embryogenesis revealed that KIP2 mRNA displays a striking pattern of expression during development, showing high level expression in skeletal muscle, brain, heart, lungs, and eye. Most of the KIP2-expressing cells are terminally differentiated, suggesting that p57KIP2 is involved in decisions to exit the cell cycle during development and differentiation. Human KIP2 is located at 11p15.5, a region implicated in both sporadic cancers and Beckwith-Wiedemann syndrome, a familial cancer syndrome, marking it as a candidate tumor suppressor. The discovery of a new member of the p21CIP1 inhibitor family with novel structural features and expression patterns suggests a complex role for these proteins in cell cycle control and development.

A plasmid carrying the 5' flanking region of the mouse proliferating-cell-nuclear-antigen (PCNA) gene or DNA polymerase beta gene was fused with the chloramphenicol acetyltransferase (CAT) gene, then cotransfected into mouse N18TG2 cells with the expression plasmid for the p53 gene. Expression of the wild-type p53 repressed the CAT expression directed by the PCNA gene promoter, while it had little effect on the DNA polymerase beta gene promoter. RNase protection analysis revealed that the repression of the PCNA gene promoter by p53 was at the transcription step. Analysis with various deletion mutants in the PCNA gene promoter revealed that a specific sequence is not required for the repression, suggesting that p53 represses the PCNA gene promoter by interacting with some components of the basic transcription machinery. By analysis with various deletion mutants in the DNA polymerase beta gene promoter, we identified the unique 10-bp palindromic sequence (-24 to -15), in the presence of which p53 was not able to repress the promoter activity. This sequence conferred resistance to p53 repression onto the PCNA gene promoter, when it was placed 21-bp upstream from the transcription-initiation site.

Different portions of the 5'-upstream region of the mouse proliferating cell-nuclear-antigen (PCNA) gene were combined with the bacterial chloramphenicol acetyltransferase (CAT) gene of a CAT vector. A transient expression assay of CAT activity in mouse neuroblastoma N18TG2 cells transfected with these recombinant plasmids and RNase protection analysis have revealed the existence of a negative regulatory region between nucleotides -1231 and -624 (+1 denotes the transcription initiation site). The CAT expression levels were gradually increased, depending on the extent of deletion from the 5'-terminus in this region, suggesting that the negative regulatory region consists of multiple elements with rather weak repressing activities. Significant sequence similarity was found between the negative regulatory region of the PCNA gene and those of the several reported genes. A 752-bp segment containing this negative regulatory region repressed the function of the PCNA gene promoter in an orientation-independent and position-independent manner. However, the negative regulatory region showed almost no repressing effect on the functions of the heterologous gene promoters such as the simian virus 40 enhancer promoter, the enhancer promoter in the Rous sarcoma virus long-terminal repeat and the mouse DNA polymerase beta gene promoter. These results suggest that the negative regulatory region of the mouse PCNA gene functions specifically to its own promoter. This unique property is discussed in comparison with that of the negative regulatory elements of the mouse DNA polymerase beta gene.

Volatile anesthetics isoflurane possibly improves the ischemic brain injury. However, its molecular actions are still unclear. In ischemia, protein kinase C (PKC)gamma and calcium/calmodulin dependent protein kinase II (CaMKII)-alpha are persistently translocated from cytosol to cell membranes, and diminish these translocation suggested to be neuroprotective. We thus tested a hypothesis that isoflurane inhibits PKCgamma and CaMKII-alpha translocation after ischemic brain insults. C57Bl/6J male mice were made to inhale 1 or 2 MAC isoflurane, after which 3 or 5 min cerebral ischemia was induced by decapitation. The sampled cerebrum cortex was then homogenized and centrifuged into crude synaptosomal fractions (P2), cytosolic fractions (S3), and particulate fractions (P3). CaMKII-alpha and PKCgamma levels of these fractions were analyzed by immunoblotting. PKCgamma and CaMKII-alpha are translocated to synaptic membrane from cytosol by cerebral ischemia, although isoflurane significantly inhibited such translocation. These results may explain in part the cellular and molecular mechanisms of neuroprotective effects of isoflurane.

Cellular responses to DNA damage are mediated by a number of protein kinases, including ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related). The outlines of the signal transduction portion of this pathway are known, but little is known about the physiological scope of the DNA damage response (DDR). We performed a large-scale proteomic analysis of proteins phosphorylated in response to DNA damage on consensus sites recognized by ATM and ATR and identified more than 900 regulated phosphorylation sites encompassing over 700 proteins. Functional analysis of a subset of this data set indicated that this list is highly enriched for proteins involved in the DDR. This set of proteins is highly interconnected, and we identified a large number of protein modules and networks not previously linked to the DDR. This database paints a much broader landscape for the DDR than was previously appreciated and opens new avenues of investigation into the responses to DNA damage in mammals.

53BP1 binds to the tumor suppressor protein p53 and has a potential role in DNA damage responses. We used small interfering RNA (siRNA) directed against 53BP1 in mammalian cells to demonstrate that 53BP1 is a key transducer of the DNA damage checkpoint signal. 53BP1 was required for p53 accumulation, G 2 -M checkpoint arrest, and the intra-S-phase checkpoint in response to ionizing radiation. 53BP1 played a partially redundant role in phosphorylation of the downstream checkpoint effector proteins Brca1 and Chk2 but was required for the formation of Brca1 foci in a hierarchical branched pathway for the recruitment of repair and signaling proteins to sites of DNA damage.

The protein kinase Chk2, the mammalian homolog of the budding yeast Rad53 and fission yeast Cds1 checkpoint kinases, is phosphorylated and activated in response to DNA damage by ionizing radiation (IR), UV irradiation, and replication blocks by hydroxyurea (HU). Phosphorylation and activation of Chk2 are ataxia telangiectasia-mutated (ATM) dependent in response to IR, whereas Chk2 phosphorylation is ATM-independent when cells are exposed to UV or HU. Here we show that in vitro , ATM phosphorylates the Ser-Gln/Thr-Gln (SQ/TQ) cluster domain (SCD) on Chk2, which contains seven SQ/TQ motifs, and Thr68 is the major in vitro phosphorylation site by ATM. ATM- and Rad3-related also phosphorylates Thr68 in addition to Thr26 and Ser50, which are not phosphorylated to a significant extent by ATM in vitro . In vivo , Thr68 is phosphorylated in an ATM-dependent manner in response to IR, but not in response to UV or HU. Substitution of Thr68 with Ala reduced the extent of phosphorylation and activation of Chk2 in response to IR, and mutation of all seven SQ/TQ motifs blocked all phosphorylation and activation of Chk2 after IR. These results suggest that in vivo , Chk2 is directly phosphorylated by ATM in response to IR and that Chk2 is regulated by phosphorylation of the SCD.

In response to DNA damage and replication blocks, cells prevent cell cycle progression through the control of critical cell cycle regulators. We identified Chk2, the mammalian homolog of the Saccharomyces cerevisiae Rad53 and Schizosaccharomyces pombe Cds1 protein kinases required for the DNA damage and replication checkpoints. Chk2 was rapidly phosphorylated and activated in response to replication blocks and DNA damage; the response to DNA damage occurred in an ataxia telangiectasia mutated (ATM)-dependent manner. In vitro, Chk2 phosphorylated Cdc25C on serine-216, a site known to be involved in negative regulation of Cdc25C. This is the same site phosphorylated by the protein kinase Chk1, which suggests that, in response to DNA damage and DNA replicational stress, Chk1 and Chk2 may phosphorylate Cdc25C to prevent entry into mitosis.

Parental origin-specific alterations of chromosome 11p15 in human cancer suggest the involvement of one or more maternally expressed imprinted genes involved in embryonal tumor suppression and the cancer-predisposing Beckwith-Wiedemann syndrome (BWS). The gene encoding cyclin-dependent kinase inhibitor p57KIP2, whose overexpression causes G1 phase arrest, was recently cloned and mapped to this band. We find that the p57KIP2 gene is imprinted, with preferential expression of the maternal allele. However, the imprint is not absolute, as the paternal allele is also expressed at low levels in most tissues, and at levels comparable to the maternal allele in fetal brain and some embryonal tumors. The biochemical function, chromosomal location, and imprinting of the p57KIP2 gene match the properties predicted for a tumor suppressor gene at 11p15.5. However, as the p57KIP2 gene is 500 kb centromeric to the gene encoding insulin-like growth factor 2, it is likely to be part of a large domain containing other imprinted genes. Thus, loss of heterozygosity or loss of imprinting might simultaneously affect several genes at this locus that together contribute to tumor and/or growth- suppressing functions that are disrupted in BWS and embryonal tumors.

A plasmid carrying the 5′-flanking region (– 1584 to + 47 with respect to the transcription initiation site) of the mouse proliferating cell nuclear antigen (PCNA) gene was fused with the chloramphenicol acetyltransferase (CAT) gene, and then cotransfected into mouse N18TG2 cells with expression plasmids for the adenovirus type 12 E1 genes. Expression of E1A gene products elevated the CAT expression by 5- to 9-fold, but expression of the E1B gene product did not. RNase protection analysis revealed that the activation of the PCNA gene promoter by E1A was at the transcription step. Both the 13S E1A and the 12S E1A activated the PCNA gene promoter, indicating that the activation domain of El A resides in a common region(s) of 13S and 12S El A products. The major target region of El A was mapped within the 68 base-pair region (-21 to +47) of the PCNA gene, which includes consensus sequences for transcription factors PEA3 and E2P, although the upstream region (–83 to – 21) including ATF(CREB)-binding consensus had an additional effect in the transactivation.

We have isolated clones containing the entire mouse proliferating cell nuclear antigen (PCNA) gene of 3890 bp and flanking sequences using a rat PCNA cDNA as a probe. The mouse gene has 6 exons whose sequences and junction points of exons with introns are extensively homologous to the human gene while sizes and nucleotide sequences of introns are much less conserved than exons. By a transient expression assay of chloramphenicol acetyltransferase, the promoter of this gene is localized within 200 bp upstream of the transcription initiation site. We have also isolated two processed pseudogenes. Homology between the first one (psi PCNA-I) and the exons of the PCNA gene was 76.8% in the region so far sequenced. The second one (psi PCNA-II) consists of a region highly homologous to the entire exons of the PCNA gene, and only 9 out of total 1256 bp are different from the corresponding exon sequence of the gene. The 5'-flanking region of the psi PCNA-II did not function as an active promoter. Surveys in various wild and laboratory mice genomes suggest that the psi PCNA-II was generated through the reverse transcription process of the PCNA mRNA about 5 x 10(5) years ago in the domesticus subspecies of Mus musculus, the house mouse. The psi PCNA-II is tentatively mapped in the chromosome 17 of the C57BL mouse.

Chk2 is a protein kinase that is activated in response to DNA damage and may regulate cell cycle arrest. We generated Chk2-deficient mouse cells by gene targeting. Chk2−/−embryonic stem cells failed to maintain γ-irradiation–induced arrest in the G2phase of the cell cycle. Chk2−/−thymocytes were resistant to DNA damage–induced apoptosis. Chk2−/−cells were defective for p53 stabilization and for induction of p53-dependent transcripts such as p21 in response to γ irradiation. Reintroduction of the Chk2 gene restored p53-dependent transcription in response to γ irradiation. Chk2 directly phosphorylated p53 on serine 20, which is known to interfere with Mdm2 binding. This provides a mechanism for increased stability of p53 by prevention of ubiquitination in response to DNA damage.